Extraction and Characterization of Tau Protein

Slices of tissue weighing 200–350 mg were first homogenized to a final 15% (w/v) concentration in calcium-free and magnesium-free PBS, pH 7.4, by 3 × 75 s cycles with a Mini-Beadbeater 16 Cell Disrupter (Biospec). Homogenates were then diluted to a final 10% (w/v) in 1% (v/v) sarkosyl in PBS, pH 7.4 and re-homogenized. After clarification at 500×g for 5 min, additional sarkosyl was added to a final 2% and the samples were spun at 15,500 rpm at 4 °C for 30 min in Allegra X-22R tabletop centrifuge (Beckman Coulter). To investigate protease-sensitive and resistant fractions of tau, the sample aliquots were digested with Proteinase K (PK) according the rodent and human prion protocol at 1:50 enzyme/total protein (weight/weight) ratio [50 (link), 85 (link), 87 (link)]. The pellet containing sarkosyl-insoluble tau protein was resuspended in PBS, pH 7.4 and divided into two aliquots: the first one was incubated with 100 µg/ml of PK (Amresco) for 1 h at 37 °C with shaking of 600 rpm in an Eppendorf Thermomixer (Eppendorf) and the second one, untreated, was mixed with protease inhibitors cocktail (0.5 mM PMSF and aprotinin and leupeptin at 5 µg/ml, respectively). After blocking PK-treated aliquots with a protease inhibitor cocktail, samples were stored for analysis at − 80 °C.